RU2754453C1 - Biocompatibility-ensuring coating for continuous analyte measurement - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: proposed is a biosensor for determining an analyte, a method for manufacturing an implantable biosensor, an application of a biocompatibility-ensuring layer, and a method for continuous determination of an analyte in the body of a subject. The biosensor is coated with a biocompatibility-ensuring layer. Said layer comprises the polymer comprising side groups -CO-NR1R2, wherein R1and R2are independently selected from -H and C1-C6-alkyl, and has the chemical structure (II) or (IIa):wherein R4constitutes a crosslinkable or crosslinked side group, n is in the range from 0.01 to 0.99, and m is in the range from 0.99 to 0.01. The method for manufacturing includes partially coating the biosensor with said biocompatibility-ensuring layer. The method for determining an analyte includes determining the analyte by means of said biosensor.EFFECT: inventions ensure elimination of fouling and encapsulation of the implantable biosensors.14 cl, 5 dwg, 4 ex

Description

The technical field to which the invention relates

The present invention relates to a biosensor for determining an analyte comprising a sensor module at least partially coated with a biocompatible layer, the biocompatible layer comprising a polymer having side groups —CO — NR ¹ R ² , where R ¹ and R ^{2 are} independently selected from - H and C ₁ -C ₆ -alkyl. In addition, the present invention relates to a method for manufacturing said biosensor, as well as applications and methods of use related to said biosensor.

State of the art

Biosensors for measuring analytes in biological fluids, in particular sensors designed for implantation, must perform a number of functions: on the one hand, the sensor must ensure the specificity and sensitivity of the measurement, excluding the influence of interferences arising, for example, from certain compounds, such as cells contained in physiological fluids. For this purpose, biosensors are often coated with membranes that trap certain compounds in order to provide access to the sensor module proper only for low-molecular-weight compounds, i.e. compounds with low molecular weight. On the other hand, for implantable sensors, it is preferable that such sensors can remain in place for a long time without degrading the measurement quality, in order to save the patient from the need for frequent sensor replacement. Accordingly, the implanted sensors are coated with biocompatible polymers to avoid the body's perception of the implanted sensor as a foreign body and sensor rejection. In addition, a suitable biocompatible layer should prevent the deposition of constituent components of physiological fluids, for example, polypeptides or cells, on the membrane, since this leads to clogging of the pores of the membrane, which causes an increase in the measurement noise and a decrease in the intensity of the useful signal.

In particular, it was noticed that a layer of cells is often formed on the implanted biosensors, which prevents the passage of molecules to the sensor, which are unable to easily diffuse through the layer of cells, including, for example, glucose molecules. Accordingly, it was proposed to provide a membrane that interferes with the formation of a barrier cell layer, and to protect the sensitive areas of the implantable device from biofouling due to the inclusion of bioactive agents in the biointerface membrane (US 2006/0198864 A1). In addition, biocompatible polymers such as ADAPT ™ formulations are used to improve the performance of biosensors. Hu and colleagues have developed an enzyme-based antifouling zwitterionic coating for biosensors (Hu et al., Langmuir, 2016, 32, 11763-11770) based on pSBMA (poly-N- (3-sulfopropyl) -N- (methacryloxyethyl) -N, N-dimethylammonium betaine) using electrochemically induced atom transfer radical polymerization (eATRP). Krishnan and colleagues (Krishnan et al., J. Mater. Chem., 2008, 18, 3405-3413) discuss a number of anti-biofouling polymers, including hydrophilic pegylated polymers, zwitterionic polymers and polymers containing oligosaccharide groups, mainly in the context of marine coatings.

However, biosensors such as implantable biosensors for continuous glycemic monitoring (LMWH) with currently used biocompatible polymers can begin to show signs of perceptible fouling after a relatively short time of wearing.

The problem to be solved

Therefore, the present invention was based on the task of providing improved means and methods for coating biosensors, allowing at least partially to eliminate the disadvantages of the prior art, in particular the fouling and encapsulation processes.

Disclosure of the essence of the invention

This problem is solved in the proposed in the present invention means and methods, characterized by the features of the independent claims. Preferred embodiments of the invention, which may be carried out individually or in any combination, are indicated in the dependent claims.

Accordingly, the present invention relates to an analyte sensor comprising a sensor module at least partially provided with a biocompatible coating, the biocompatible coating comprising a polymer having side groups —CO — NR ¹ R ² , wherein R ¹ and R ^{2 are} independently selected from -H and C ₁ -C ₆ -alkyl.

In the text of the description and the claims, the terms "has", "contains", "includes (includes)" or any of their grammatical variations are used in a non-exclusive manner, leaving the corresponding wording open. Thus, these terms can be used both in a situation in which in the corresponding object in the context of the invention there are no other features other than the feature introduced by these terms, and in a situation in which one or more other features are also present. For example, the expressions "A has B", "A contains B" and "A includes B" can be used as in a situation in which there are no other elements in object A besides B (i.e. in a situation in which A consists of B and only B), and in a situation in which object A, in addition to B, contains one or more other elements, for example, element C, elements D and D, or other additional elements.

Further, below in the text, the expressions "preferably", "more preferably", "especially", "predominantly", "in particular", "primarily" or similar expressions are used in relation to optional features, without limiting other possibilities. Therefore, the features introduced by these expressions are optional, i.e. are optional and are not intended to limit the scope of the patent claims in any way. As should be understood by a person skilled in the art, implementation of the invention is possible using alternative features. Likewise, the features introduced by the expression "in one embodiment (implementation of the invention)" or similar expressions are intended to be optional and do not imply any limitation with respect to other embodiments of the invention, with respect to the scope of the invention, and with respect to the possibilities of combining introduced thus features with other optional or mandatory features of the invention.

In the context of the invention, the term "standard conditions", unless otherwise indicated, refers to the standard IUPAC conditions with respect to ambient temperature and pressure, ie, in one embodiment, it refers to a temperature of 25 ° C and an absolute pressure of 100 kPa; also in one embodiment, standard conditions include pH = 7. In addition, unless otherwise indicated, the words "about", "about" or "about" preceding the indication of a value means the applicability of technical tolerances for the indicated value generally accepted in the relevant field of technology, and in one embodiment of the invention these words indicate that the tolerances for said value are ± 20%, in yet another embodiment, the tolerances are ± 10%, in yet another embodiment, the tolerances are ± 5%. In addition, the term "substantially" indicates that there is no variation affecting the specified result or application, i. E. potential deviations do not lead to the fact that the specified result deviates by more than ± 20%, in another embodiment of the invention by more than ± 10%, in another embodiment of the invention by more than ± 5%. Thus, the expression "consisting essentially of" means the inclusion in the composition of the components indicated in this expression and the exclusion of other components, in addition to materials present in the form of impurities, materials inevitably present as a result of the processes used to obtain these components, and the components added with a purpose other than the achievement of the technical result of the invention. For example, a composition as defined using the phrase "consisting essentially of" includes any known acceptable additive, filler, diluent, carrier, and the like. In one embodiment of the invention, a composition consisting essentially of a set of components will contain an unspecified component (s) in this set in an amount of less than 5 wt. %, in another embodiment of the invention in an amount of less than 3 wt. %, in another embodiment of the invention in an amount of less than 1%, in another embodiment of the invention in an amount of less than 0.1 wt. %.

The term "biosensor" in the context of the invention refers to a device containing the means described in this description. The biosensor contains at least a sensor module and a biocompatible layer, which are described below. In one embodiment of the invention, the biosensor is implantable, in particular subcutaneously implantable. In one embodiment, the biosensor is fully implantable, i. E. the biosensor is implanted together with its external communication means, i.e. communication with external devices, while, in particular, for the operation of the biosensor does not require a hole in the skin of the subject. Thus, in one embodiment of the invention, the interaction of the biosensor with external devices is carried out via wireless communication. As will be appreciated by one of ordinary skill in the art, insertion of the biosensor into the body may require an opening in the skin of a subject.

In one embodiment, the sensor also has a window, i. E. an opening allowing the sensor module to contact the environment, in particular physiological fluid. In one embodiment of the invention, said window is located near the sensor module, and a biocompatible layer, optionally in combination with a diffusion membrane, separates the sensor module from the medium to be analyzed. In addition, in the particular case when the biosensor is implantable, it also contains electrical and electronic means, including, for example, an energy source, such as a battery, and / or a communication unit for exchanging information with an external device, for example, for transmitting analyte values ... Means suitable for realizing energy sources and communication units are well known in the art. In one embodiment of the invention, the biosensor also comprises a processing unit that converts the detected signal, or a physical signal derived therefrom, such as an electrical signal, into a signal transmitted to a reading unit, such as a reader. In yet another embodiment of the invention, the processing unit converts the detected signal or a physical signal derived therefrom into a measured value (measurement result), for example, an analyte concentration. It should be borne in mind that the aforementioned means may perform additional functions, and that the biosensor may contain other means that are relevant from the point of view of a specialist.

In one embodiment, the biosensor also comprises a diffusion membrane. The term "diffusion membrane" in the context of the invention refers to a polymer that allows diffusion of an analyte, i. E. allowing the analyte to diffuse through itself, in particular refers to a polymer with a thickness of 1 to 100 μm, in particular a thickness of 2.5 to 50 μm. In one embodiment, the diffusion membrane comprises a biocompatible polymer, in particular a polymer obtained by polymerizing butyl methacrylate (BUMA) and / or 2-hydroxyethyl methacrylate (HEMA) monomers, or consists of such a biocompatible polymer. In yet another embodiment of the invention, the diffusion membrane comprises a hydrophilic polyurethane polymer (HPU). In yet another embodiment of the invention, the diffusion membrane permits the diffusion of the analyte, but not of the high molecular weight components contained in the medium to be analyzed, for example in physiological fluid. Accordingly, in one embodiment of the invention, the diffusion membrane, in particular, allows the diffusion of molecules with a molecular weight of less than 10 kDa, in particular less than 5 kDa, in particular less than 1 kDa. Thus, in one embodiment of the invention, the diffusion membrane is a semipermeable membrane, in particular a dialysis membrane, in particular a biocompatible dialysis membrane.

In one embodiment, the biosensor is completely coated with a biocompatible layer. In yet another embodiment of the invention, the sensor is made of an inert material and comprises a window as described above, such window being at least partially covered with a biocompatible layer; or the window and / or sensor module is at least partially covered with at least a biocompatible layer, wherein the expression "at least partially covered" means that at least a sufficient surface area is covered with the biocompatible layer to perform an analyte measurement. In one embodiment, the biocompatible membrane covers at least 25%, in another embodiment at least 50%, in another embodiment at least 75%, in another embodiment at least 90% of the window area and / or the test field of the sensor module, as indicated below.

In one embodiment, the biosensor is an electrochemical biosensor; thus, the biosensor can contain additional electrical leads and contacts. In one embodiment of the invention, the biosensor contains additional electrodes, which can be electrodes having the characteristics described below, or electrodes that are structurally and / or functionally different from them. Additional electrodes can be adapted, for example, for use in filling control, as a temperature sensor, etc. In one embodiment of the invention, the biosensor also comprises a counter electrode, in another embodiment of the invention also comprises a reference electrode and a counter electrode. It is known from the prior art that a biosensor may contain three electrodes, each of which corresponds to a working (indicator) electrode, a counter electrode and a reference electrode ("three-electrode circuit"); or the biosensor may contain two electrodes, one of which is a working electrode and the other is a counter electrode and a reference electrode ("two-electrode circuit"). As indicated above, the biosensor can also contain additional electrodes. In the context of the present invention, at least the working electrode is in electrical, i. E. conducting, contact with at least a portion of the test field.

In one embodiment, the biosensor is an optical biosensor; thus, the biosensor may contain additional optical means, for example an illumination unit and / or an optical radiation-sensitive (light-sensitive) element, for example a photocell. In one embodiment, the sensor module comprises at least one light source for illuminating at least a portion of the test field discussed herein, and comprises at least one optical radiation sensitive element capable of detecting detection (response) light emanating from the specified test field. Thus, in one embodiment of the invention, the sensor module comprises at least one light source, such as a light emitting diode (LED). In one embodiment of the invention, the aforementioned detection (response) light is selected from the group consisting of light reflected by the test field, light transmitted by the test field, and light emitted by the test field. In one embodiment of the invention, the optical radiation sensitive element includes at least one element capable of detecting light emitted by the light source and reflected and / or transmitted by the test field. The specified light-sensitive element can be, for example, a photodiode. In one embodiment of the invention, the light-sensitive element includes at least one one-dimensional or two-dimensional array of light-sensitive elements, in one embodiment of the invention at least one camera chip, in particular at least one chip on charge devices. communication (CCD). In one embodiment of the invention, the optical signal is converted into an electrical signal after registration, and thus the biosensor may contain additional elements described above for the electrochemical sensor and relevant from the point of view of a specialist.

The term "sensor module" in the context of the invention refers to a device comprising at least one test field, generating a detectable signal in the presence of an analyte, and optionally also containing a signal detection unit that detects and / or converts the detected signal into an output signal, for example an electrical output. signal, such as current, voltage, etc. In one embodiment of the invention, the sensor module is selected from an optical sensor module and an electrochemical sensor module as described above. In principle, both of these types of sensor modules are known from the prior art.

The term "detectable signal" in the context of the invention refers to a property of an analytical (indicator) reagent that changes in the presence of an analyte and which can be converted into a physical signal of any kind. In one embodiment of the invention, the change in this measured property and / or the signal generated therefrom is proportional to the concentration of the analyte in the sample. In one embodiment of the invention, as described above, the measured property is a change in color and / or color intensity of an analytical reagent, i. E. in particular, a change in the absorption and / or emission spectrum of an analytical reagent. Thus, when the measured property changes, the optical property in one embodiment of the invention is selected from the group consisting of: reflection property, in particular reflectivity and / or diffuse reflection; transmission properties, in particular absorption; colors; luminescence, in particular fluorescence. Also in one embodiment, the property being measured is the concentration of a reduced or oxidized redox compound, eg, a mediator, as discussed herein. Methods for converting a measurable property defined above into a physical signal that can be read as a measured value are well known in the art and are described, for example, in EP 0821234, EP 0974303 and US 2005/0023152.

In one embodiment of the invention, the property being measured is the redox state of a redox compound contained in the analytical reagent and is detected by electrochemical means, for example, by measuring voltage, capacitance, admittance, current, or any parameter relevant to of a specialist's point of view. Thus, the present invention also provides that the analytical reagent includes a chemical reagent for reacting with the analyte to generate an electrochemical signal indicative of the presence of the analyte in a liquid sample. In the case of glucose as the preferred analyte, the active components of the analytical reagent typically include an enzyme using, in particular specifically using, glucose, and optionally a redox mediator. In another embodiment, said enzyme comprises glucose oxidase and / or glucose dehydrogenase. In one embodiment, the enzyme, in the presence of the analyte, produces a reduced cofactor, for example NAD (P) H, and the mediator, in turn, reacts with the reduced cofactor. The mediator then transfers the redox equivalent of the analyte product to the electrode surface by diffusion. There, the mediator is oxidized quantitatively at a certain anodic potential, and the resulting current is related to the apparent glucose concentration. Preferred redox mediators are generally rapidly reducing and oxidizing molecules. Examples include ferricyanide, nitrosoaniline and their derivatives, as well as ferrocene and its derivatives. In yet another embodiment of the invention, an enzyme, for example glucose oxidase, produces a peroxide, in particular hydrogen peroxide, which is determined electrochemically. There are a number of reagent systems suitable for glucose determination, examples of which include solutions for AC excitation, analyte sensors and biosensors described in US patents 5385846, US5997817 and in application No. with an electrochemical biosensor "); reagent KNAD (carbANAD) described in publications WO 2007/012494, WO 2009/103540, WO 2011/012269, WO 2011/012270 and WO 2011/012271, and SCV reagent described in publications EP 0354441, EP 0431456.

In yet another embodiment of the invention, the analytical reaction is accompanied by a color change of the analytical reagent, or at least part of it. In one embodiment of the invention, the analytical reagent comprises at least one enzyme which, in particular directly or indirectly, reacts with the analyte, in particular reacts with high specificity, wherein one or more optical indicator substances are also present in the analytical reagent, which, when the reactions of the at least one enzyme with the analyte indicate at least one change in the optically detectable property. Thus, at least one indicator may include one or more dyes participating in a coloring reaction indicating an enzymatic reaction of at least one enzyme and analyte. An example of the optical determination of an analyte is disclosed, for example, in WO 2015/005953 A1.

The term "test field" in the context of the invention refers to a continuous (coherent) or discontinuous (non-coherent) amount of an analytical reagent, which, in particular, is located on at least one carrier, for example, on at least one film carrier. Thus, an analytical reagent considered elsewhere in the description may form one or more films or layers of the test field, or may be contained in such film (s) or such layer (s) of the test field, and / or the test field may comprise a layered structure with one or more layers, in which at least one of the layers contains an analytical reagent. Thus, the test field can contain a layered structure located on the carrier, and the sample can be in contact with the layered structure from at least one side of the sample application. In one embodiment of the invention, the test field has a multilayer structure comprising at least one assay layer having at least one assay reagent and also comprising at least one separation layer capable of separating at least one particulate component contained in physiological fluid, and the separating layer is located between the analytical layer and the sample access site.

The terms "analytical reagent" or "analytical material" refer to a substance or mixture of substances that, in the presence of an analyte, is (are) capable of causing a change in at least one measurable property. In one embodiment, the analyte participates in at least one optically or electrochemically detectable analytical reaction in the presence of the analyte. In one embodiment of the invention, the analytical reaction proceeds, at least in part, by means of at least one enzyme, and accordingly, in one embodiment of the invention, the analytical material comprises at least one enzyme capable of participating in at least one enzymatic reaction. As far as the analytical reagent is concerned, various possibilities for the preparation of analytical reagents are known in the art. The analytical reagent is selected according to the analyte to be determined.

и соавт.: The Technology Behind Glucose Meters: Test Strips (Технические средства глюкометров: тест-полоски), Diabetes Technology & Therapeutics, том 10, дополнение 1, 2008, стр. 10-26. Вместе с тем, могут использоваться и другие типы ферментов и/или другие типы аналитического реагента или активных компонентов аналитического реагента.The term "enzyme" in the context of the invention refers to a biological macromolecule, in particular to a polypeptide, which catalyzes, in the presence of an analyte, a chemical reaction resulting in the formation or consumption of a compound providing a detectable signal. In one embodiment of the invention, the enzyme is an oxidoreductase, in particular an oxidase; in yet another embodiment of the invention, the enzyme produces, in the presence of an analyte, peroxide, in particular hydrogen peroxide; thus, in one embodiment, the enzyme is a hydrogen peroxide-producing oxidase, for example glucose oxidase (EC 1.1.3.4), hexose oxidase (EC 1.1.3.5), cholesterol oxidase (EC 1.1.3.6), galactose oxidase (EC 1.1.3.9), alcohol oxidase (EC 1.1.3.13), L-gulonolactone oxidase (EC 1.1.3.8), NAD (P) H-oxidase (forming Н ₂ О ₂ , EC 1.6.3.1), NADH-oxidase (forming Н ₂ О ₂ , EC 1.6. 3.3) or catalase (EC 1.11.1.6). In one embodiment of the invention, considered elsewhere in the description, the analyte is glucose. Thus, at least one enzyme may include an enzyme, in particular an oxidoreductase, having glucose as a substrate, in particular, it may include glucose oxidase and / or glucose dehydrogenase. In another embodiment, the enzyme is glucose oxidase (EC 1.1.3.4). In this regard, reference can be made to the aforementioned prior art documents. In particular, you can refer to the work

et al: The Technology Behind Glucose Meters: Test Strips, Diabetes Technology & Therapeutics, Volume 10, Supplement 1, 2008, pp. 10-26. However, other types of enzymes and / or other types of analytical reagent or active components of an analytical reagent can be used.

The term "biocompatible layer" in the context of the invention refers to a layer, in particular an outermost layer, of a biosensor or part thereof, consisting of a biocompatible material. In one embodiment, the biocompatibility layer has a thickness of 5 nm to 100 μm, in particular 10 nm to 1 μm. In one embodiment of the invention, the biocompatible layer at least partially covers the test field of the sensor module, in another embodiment of the invention at least partially covers the sensor module, in particular completely covers the areas of the sensor module that are not in contact with other elements of the biosensor, especially at least partially or completely covers the biosensor. In one embodiment, the biocompatible layer is the outermost layer of the sensor module and / or biosensor. Thus, in one embodiment of the invention, at least a portion of the biocompatibility layer is in contact with the physiological fluid of the subject. In one embodiment, the biocompatible layer does not restrict diffusion for the analyte as discussed elsewhere in the specification; in yet another embodiment of the invention, the biocompatible layer does not restrict diffusion for small molecules having a molecular weight of less than 2000 Da, in particular less than 1000 Da. In one embodiment of the invention, the biocompatibility layer contains no added enzyme, in particular no added polypeptide; as should be clear to the skilled person, this does not preclude the enzyme or polypeptide molecules diffusing into the biocompatible layer from adjacent layers, tissues, or physiological fluids.

The term "biocompatible material" in the context of the invention refers to a material that is suitable for use with living tissue or in a living system due to the fact that such material is not, or is only to a lesser extent toxic, harmful or inducing physiological reactions, and / or the fact that it causes only a diminished degree or does not cause immunological rejection. In one embodiment of the invention, the biocompatible material is a material that does not elicit a systemic response from the body, for example, an inert material or a material containing chemical compounds that prevent the occurrence of systemic responses of the body in the region of the biocompatible layer. In another embodiment of the invention, the biocompatible material is a material that prevents cells from attaching to said biocompatible layer. In another embodiment of the invention, as indicated above, the biosensor comprises at least two layers, of which the outer layer is a layer,

containing said polymer with side groups —C (O) —NR ¹ R ² , i. e. biocompatible layer. In one embodiment of the invention, the second layer is a diffusion membrane, discussed elsewhere in the description. In one embodiment of the invention, the biocompatibility layer and the diffusion membrane are covalently linked, in particular by photoactivation, by means of a cross-linkable side group, as described below.

The biocompatible layer disclosed herein comprises a polymer having side groups —CO — NR ¹ R ² , where R ¹ and R ^{2 are} independently selected from —H and C ₁ -C ₆ alkyl, in particular consisting of such a polymer. Thus, in one embodiment, said polymer contains unsubstituted amide side groups. In one embodiment, at most one of R ¹ and R ² is —H. Thus, in one embodiment, said polymer contains mono- and / or di-N-substituted amide side groups.

In the context of the invention, the term "alkyl" refers to a monovalent side group derived from an alkane by removing a hydrogen atom from any carbon atom. In one embodiment, alkyl is a straight or branched chain saturated hydrocarbon group. Further, the alkyl groups are straight chain alkyls, for example, in particular methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, or branched chain alkyl groups, for example -CH (CH ₃ ) ₂ , -CH (CH ₂ CH ₃ ) ₂ , -C (CH ₃ ) ₃ , -C (CH ₂ CH ₃ ) ₃ , -CH (CH ₃ ) (CH ₂ CH ₃ ), -CH ₂ CH (CH ₃ ) ₂ , -CH ₂ CH (CH ₃ ) (CH ₂ CH ₃ ), -CH ₂ CH (CH ₂ CH ₃ ) ₂ , -CH ₂ C (CH ₃ ) ₃ , -CH (CH ₃ ) CH (CH ₃ ) (CH ₂ CH ₃ ), -CH ₂ CH ₂ CH (CH ₃ ) ₂ , -CH ₂ CH ₂ CH (CH ₃ ) (CH ₂ CH ₃ ), -CH ₂ CH ₂ C (CH ₃ ) ₃ or -CH (CH ₃ ) CH ₂ CH (CH ₃ ) ₂ . Accordingly, alkyl groups include primary alkyl groups, secondary alkyl groups, and tertiary alkyl groups. Also contemplated are lower alkyl groups, i. E. in particular alkyl groups having at most 6 carbon atoms, especially alkyl groups having at most 3 carbon atoms, preferably alkyl groups having one carbon atom, i. e. metal groups. In one embodiment, alkyl is straight chain lower alkyl, particularly selected from n-hexyl, n-pentyl, n-butyl, n-propyl, ethyl, and methyl.

According to the invention, said polymer has a chemical structure represented by formula (II) or (IIa):

where R ⁴ is a cross-linkable side group, n is from 0.01 to 0.99, am is from 0.99 to 0.01, in particular the sum of m and n is 1. In one embodiment, n is from 0.5 to 0.99, m is from 0.5 to 0.01, and the sum of m and n is 1. As should be understood by a person skilled in the art, the sum of m and n can be less than 1 if the polymer contains other side chains or side groups; thus, in one embodiment, the sum of m plus n is at most 1; in one embodiment, the sum of m plus n is 1, i. e. the polymer only has pendant groups as shown in formula (II). In yet another embodiment, the polymer has pendant groups as shown in formula (IIa). In addition, as will be understood by a person skilled in the art, the cross ^{-linked side group R 4} can also be a cross-linked side group, i. E. can be a cross-linkable side group after cross-linking to form a covalent bond with another polymer molecule or with a diffusion membrane.

In one embodiment, R ¹ and R ^{2 are} independently selected from —H and C ₁ -C ₃ alkyl. In yet another embodiment, R ¹ and R ^{2 are} independently selected from —H, ethyl and methyl, in particular at least one of R ¹ and R ² is methyl, in particular both R ¹ and R ² are methyl.

In one embodiment, the polymer having pendant —CO — NR ¹ R ² groups is or is derived from a composition containing monomers as defined below and referred to herein as a “monomer composition”. In one embodiment, the polymer having pendant —CO — NR ¹ R ² groups is or is derived from a composition comprising monomers having the general structure (I):

where R ³ is a polymerization group. Suitable polymerizable chemical groups and methods for polymerizing them are known in the art. In one embodiment, R ^{3 is} selected from a C ₂ -C ₄ alkenyl group.

In the context of the invention, the term "alkenyl" refers to a hydrocarbon group formed by the removal of hydrogen from an alkene. In one embodiment, R ^{3 is} selected from vinyl (ie, ethenyl, —CH = CH ₂ ), propenyl (—CH = CH — CH ₃ ), and isopropenyl (—C (CH ₃ ) = CH ₂ ). In yet another embodiment, R ³ is vinyl; thus, in one embodiment, the polymer having pendant —CO — NR ¹ R ² groups is or is derived from a monomer composition containing acrylamide monomers, in particular containing monomers of mono- and / or di-N-substituted acrylamide.

In one embodiment, the monomer composition comprises a plurality of dissimilar monomers having a general structure (I) as defined above, eg at least two dissimilar monomers having a common structure (I). In one embodiment, the monomer composition comprises acrylamide monomers and at least one type of monomer of mono- and / or di-N-substituted acrylamide; or the monomer composition contains at least one type of mono- and / or di-N-substituted monomer, where R ¹ and / or R ² is (are) C ₁ -C ₃ -alkyl, in an embodiment of the invention that improves biocompatibility polymer, and at least one type of mono- and / or di-N-substituted monomer, where R ¹ and / or R ² is (are) C ₄ -C ₆ -alkyl, in an embodiment of the invention that improves mechanical properties polymer; or the monomer composition contains at least one type of mono- and / or di-N-substituted monomer, where R ¹ and R ² are C ₁ -C ₃ -alkyl, and at least one type of mono- and / or di- N-substituted monomer, where R ¹ and R ² are C ₄ -C ₆ -alkyl.

In one embodiment, the monomer composition described herein also contains at least one other monomer that does not correspond to the general structure (I). In one embodiment, such a monomer is a monomer suitable for cross-linking and / or branching of polymer chains. Both of these types of monomers are known in the art. Thus, in one embodiment of the invention, the monomer composition also contains at least one monomer having a cross-linkable side group.

In the context of the invention, the term "crosslinkable side group" refers to a chemically reactive or activatable group suitable for establishing a covalent bond from a molecule containing said crosslinkable side group to another chemical molecule or to another part of the same molecule. In one embodiment of the invention, such cross-linking is established with another polymer molecule or with a diffusion membrane discussed elsewhere in the description. As is known in the art, crosslinking can be achieved by including a side group in the polymer capable of reacting with a reactive group, or by including a chemically reactive or activatable side group in the polymer. In one embodiment, the cross-linkable group is a pendant group including the —OH or —NH ₂ group contained in a polymer that can be cross-linked with a multifunctional, in particular bifunctional, cross-linking agent known in the art, including, for example, diglycidyl ethers such as poly (ethylene glycol) diglycidyl ether and poly (propylene glycol) diglycidyl ether, bis-aldehydes and succinimidyl ethers. In yet another embodiment of the invention, the cross-linkable side group is an activatable side group, i. E. a side group that is substantially chemically unreactive under standard conditions and / or in the dark, but becomes chemically reactive upon activation, for example when illuminated with light of the appropriate wavelength. In one embodiment, the cross-linkable side group is selected from the list consisting of benzophenones, arylazides, azido-methylcoumarins, anthraquinones, certain diazo compounds, diazirines, and psoralen derivatives. In yet another embodiment, the cross-linkable side group is a benzophenone side group. In yet another embodiment, the crosslinkable side group is derived from or derived from O-substituted methacrylate monomers, in particular from 4-benzoylphenylmethacrylate monomers.

In one embodiment, the polymer having pendant —CO — NR ¹ R ² groups is or is derived from a composition containing mono-N- or di-N-substituted acrylamide monomers, in particular containing N, N-dimethylacrylamide monomers. In another embodiment of the invention, the polymer having pendant groups —CO — NR ¹ R ² is a copolymer, in particular a random copolymer. In other embodiments, such a copolymer, in addition to —CO — NR ¹ R ² , contains another repeating unit that may serve an additional function such as crosslinking, improving biocompatibility or mechanical property, as indicated above. In yet another embodiment, the polymer having pendant —CO — NR ¹ R ² groups is a copolymer of mono-N- or di-N-substituted acrylamide monomers and / or O-substituted methacrylate monomers.

In one embodiment of the invention, the polymer having pendant groups —CO — NR ¹ R ² is a copolymer obtained or obtained from a composition containing monomers having the general structure (I) as defined above and monomers containing a cross-linkable pendant group , in particular a benzophenone side group. In another embodiment of the invention, the polymer having pendant groups —CO — NR ¹ R ² contains from 1 to 50 mol. %, in particular from 2 to 25 mol. %, in particular from 3 to 10 mol. %, of units derived from monomers containing a crosslinkable side group, relative to the total amount of polymer having side groups —CO — NR ¹ R ² . In one embodiment, the polymer has a chemical structure represented by formula (III), (IIIa), (IIIb) and / or (IIIc):

where R ⁵ represents another polymer molecule or a diffusion membrane molecule.

In one embodiment, the polymer has a chemical structure represented by formula (III) or (IIIa). In one embodiment, the polymer has a chemical structure represented by formula (IIIb) or (IIIc).

Thus, in one embodiment of the invention, the polymer has a chemical structure represented by formula (IV) or (IVa):

where n is from 0.01 to 0.99, am is from 0.99 to 0.01, and where R ⁶ is —CO — C ₆ H ₅ (benzoyl) or —C (OH) R ⁵ —C ₆ H ₅ , where R ⁵ is as described above, in particular, the sum of m and n is 1. In one embodiment, n is from 0.5 to 0.99, m is from 0.5 to 0, 01, and the sum of m and n is 1.

In yet another embodiment, the polymer having pendant —CO — NR ¹ R ² groups is a terpolymer of at least two dissimilar mono-N- or di-N-substituted acrylamide monomers discussed elsewhere in the description and monomers O -substituted methacrylate, in particular 4-benzoylphenylmethacrylate monomers.

The term "analyte" in the context of the invention refers to a chemical compound present in a fluid, in particular a physiological fluid. In one embodiment, the analyte is an organic molecule, in particular an organic molecule capable of undergoing a redox reaction in the presence of an enzyme within the meaning of the present invention. In one embodiment, the analyte is a molecule involved in the metabolism of a subject, i. E. a molecule formed and / or consumed in the course of at least one chemical reaction occurring in at least one tissue of the specified subject. In addition, in one embodiment of the invention, the analyte is a low molecular weight chemical, in particular a chemical with a molecular weight of less than 5000 amu. (5000 Da; 1 amu = 1.66 × 10 ^-27 kg), in particular less than 1000 amu, primarily less than 500 amu. That is, in one embodiment, the analyte is not a biological macromolecule. In yet another embodiment, said analyte is selected from the list consisting of glucose, malate, ethanol, ascorbic acid, cholesterol, glycerol, urea, 3-hydroxybutyrate, lactate, pyruvate, ketones, and creatinine; in yet another embodiment, the analyte is glucose.

The term "determination" in the context of the invention refers to the quantitative determination of an analyte in the sense of determining the values of the amount of analyte present in a physiological fluid sample, i. E. to measure the amount or concentration of the specified analyte, in particular to a semi-quantitative or quantitative measurement. Determination of the amount of analyte can be carried out in a variety of ways known to the person skilled in the art or discussed in detail elsewhere in the description. In accordance with the present invention, the determination of the amount of the analyte can be performed by any known means for determining the amount of the specified analyte in the sample. In one embodiment, the definition is a specific definition of an analyte in accordance with the present invention. In one embodiment of the invention, the determination of the analyte is the determination of the specified analyte in a living body (in vivo), in another embodiment of the invention, the determination of the analyte is continuous monitoring of glycemia, in particular the continuous monitoring of glycemia in a living body (in vivo) , first of all, the subject as defined in the description.

The term "amount" in the context of the invention includes the absolute amount of analyte, the relative amount or concentration of the analyte referred to in this description, as well as any value or parameter associated with them. Such values or parameters include measured values of intensity signals related to any particular physical or chemical properties of the analyte referred to in this description. It should be borne in mind that values related to the aforementioned quantities or parameters can also be obtained through any standard mathematical operations.

In the context of the invention, the term "physiological fluid" refers to any body fluids of a subject that are known or suspected to contain an analyte within the meaning of the present invention, including such fluids as interstitial fluid, blood, plasma, lacrimal fluid, urine , lymph, cerebrospinal fluid, bile, feces, sweat and saliva. In one embodiment, the physiological fluid is interstitial fluid or blood; thus, in one embodiment of the invention, the physiological fluid contains at least one particulate component, in particular cells. In yet another embodiment, the physiological fluid is an interstitial fluid. The term "sample" is clear to the specialist and refers to any replenishment of physiological fluid, in particular taken from the body of the subject before applying the specified sample to the test element. Samples can be obtained by commonly known techniques including, for example, vein or artery puncture, skin puncture, and the like.

In the course of work, the results which form the basis of the present invention, it was found that the described polymers are especially suitable as biocompatible coatings for biosensors. Surprisingly, it has been found that the disclosed polymers have a reduced tendency to fouling due to the accumulation of biological macromolecules and / or due to cell adhesion, have low cytotoxicity and low activity in activating the immune system. Thus, the biosensors according to the invention have an increased durability and a low tendency to encapsulate.

The above definitions apply mutatis mutandis to the following description. The additional definitions and explanations below also apply mutatis mutandis to all of the embodiments disclosed herein.

The subject of the present invention is also a method of manufacturing an implantable biosensor for analyte determination, comprising at least partially coating said biosensor with a biocompatible layer according to the present invention.

Methods for producing polymer layers and coatings on biosensors and / or sensor modules are known in the art. In this context, the coating formation process can be carried out by any method that is appropriate from the point of view of a specialist, including methods that make it possible to create a coating in situ (in situ) by applying a monomer solution to the biosensor, which is preceded, accompanied or followed by the beginning polymerization, as well as by applying a pre-fabricated membrane to the biosensor. In one embodiment of the invention, a method of making an implantable biosensor includes the use of at least one of the following methods: a dip coating method, a spray coating method, and a contact coating method. A method selected from among the methods of dip coating, spray coating, spin coating, printing coating, knife coating, or drip process can also be used. However, in principle, combinations of these methods and / or other methods can also be used. In one embodiment, a biocompatible layer is formed on the diffusion membrane. In one embodiment of the invention, the method also includes the step of cross-linking the biocompatible layer with an underlying layer, in particular a diffusion membrane, for example by irradiation, in particular by irradiation with a wavelength of 365 nm. In one embodiment, the method of making an implantable biosensor also includes the additional step of extracting unpolymerized mono- and oligomers from the biocompatible layer of the biosensor, for example by treating the biosensor with a solvent, such as an organic solvent and / or water.

The subject of the present invention is also the use of the biocompatible layer according to the present invention for the manufacture of an implantable biosensor.

The present invention also relates to the use of a biosensor according to the present invention for the continuous determination of an analyte under physiological conditions (in situ). As indicated elsewhere in the description, in one embodiment of the invention, the analyte is glucose.

The subject of the present invention is also a method for the continuous determination of an analyte in a subject's body, characterized in that the determination of said analyte is carried out by means of a biosensor according to the present invention.

In addition, the present invention relates to a method for the continuous determination of an analyte in a subject, comprising:

(a) implanting a biosensor of the present invention into at least one tissue of a subject; and

(b) determination of the specified analyte by means of the specified sensor.

Methods for the continuous determination of an analyte within the meaning of the present invention are, in one embodiment, methods carried out in vivo (in vivo). In addition, they may include steps in addition to the steps directly indicated above. For example, additional steps may relate, in particular, to the issuance of the amount or concentration of the analyte based on the determination of the specified analyte, or to the issuance of an alarm if the specified analyte determination is below the first threshold value or above the second threshold value. In addition, one or more of the above steps can be performed by automation tools. In one embodiment, the method does not include diagnosing a disease based on the result of said measurement.

In the context of the invention, the term "subject" refers to a vertebrate animal. In one embodiment, the subject is a mammal, particularly a mouse, rat, cat, dog, hamster, guinea pig, sheep, goat, pig, cow, or horse. In particular, the subject is a primate. First of all, the subject is a person. In one embodiment of the invention, the subject suffers or is suspected of suffering from a disease or condition associated with a measurable deviation of at least one analyte from the normal value. In yet another embodiment, the subject is diabetic.

The term "continuous determination" in the context of the invention refers to the determination of an analyte by repeated use in a subject's body of the same biosensor, in particular the biosensor disclosed herein. Thus, the term "continuous determination" includes discrete measurements, in particular measurements performed at least every 6 hours, in particular at least every 2 hours, in particular at least every hour. However, this term also covers more frequent measurements, in particular measurements carried out at least every 30 minutes, in particular at least every 15 minutes, in particular at least every 10 minutes, in particular at least every 5 minutes. ... In one embodiment of the invention, this term refers to a quasi-continuous measurement carried out in particular every 30 seconds, in particular every 20 seconds, in particular at least every 10 seconds, in particular at least every 5 seconds. However, the invention provides for the possibility of more frequent measurements. In one embodiment of the invention, the continuous determination of the analyte is carried out by means of the same biosensor for at least one week, in another embodiment for at least two weeks, in another embodiment for at least three weeks, in another embodiment for at least four weeks.

Based on the foregoing, the following embodiments of the invention are preferred:

1. A biosensor for determining an analyte comprising a sensor module at least partially covered with a biocompatible layer, the biocompatible layer comprising a polymer having side groups —CO — NR ¹ R ² , where R ¹ and R ^{2 are} independently selected from —H and C ₁ -C ₆ -alkyl, in particular where at most one of R ¹ and R ² is —H.

2. A biosensor according to embodiment 1, wherein R ¹ and R ^{2 are} independently selected from —H and C ₁ -C ₃ -alkyl.

3. A biosensor according to embodiment 1 or 2, wherein R ¹ and R ^{2 are} independently selected from —H, ethyl and methyl.

4. A biosensor according to one of embodiments 1-3, wherein at least one of R ¹ and R ² is methyl.

5. A biosensor according to one of embodiments 1-4, wherein R ¹ and R ² are methyl.

6. A biosensor according to one of embodiments 1-5, wherein said polymer having pendant groups —CO — NR ¹ R ² is obtained or obtained from a composition containing monomers having the general structure (I):

where R ³ is a polymerizable group, in particular selected from vinyl, propenyl and isopropenyl, in particular representing vinyl.

7. A biosensor according to one of embodiments 1-6, wherein said polymer having pendant groups —CO — NR ¹ R ² is obtained or obtained from a composition containing mono-N- and / or di-N-substituted acrylamide monomers, in particular containing N, N-dimethylacrylamide monomers.

8. A biosensor according to one of embodiments 1-7, wherein said polymer having pendant —CO — NR ¹ R ² groups is a copolymer, in particular a random copolymer.

9. A biosensor according to one of embodiments 1-8, wherein said polymer having pendant groups —CO — NR ¹ R ² is a copolymer of mono-N- or di-N-substituted acrylamide monomers and / or O-substituted methacrylate monomers ...

10. Biosensor according to embodiment 9, wherein said monomers of O-substituted methacrylate are monomers of 4-benzoylphenylmethacrylate.

11. A biosensor according to one of embodiments 1-10, wherein said polymer has a chemical structure represented by formula (II) or (IIa):

where R ⁴ is a cross-linkable or cross-linked side group, n is from 0.01 to 0.99, am is from 0.99 to 0.01, in particular, the sum of m and n is 1.

12. A biosensor according to one of embodiments 1-11, in which n is from 0.5 to 0.99, m is from 0.01 to 0.5, and the sum of m and n is 1.

13. A biosensor according to one of embodiments 1-12, wherein said polymer having pendant groups —CO — NR ¹ R ² is a copolymer obtained or obtained from a composition containing monomers described in embodiment 6 or 7 and monomers, containing a cross-linkable side group, in particular a benzophenone side group.

14. Biosensor according to one of options 1-13, in which the specified polymer having side groups -CO-NR ¹ R ² contains from 1 to 50 mol. %, in particular from 2 to 25 mol. %, in particular from 3 to 10 mol. %, of units derived from monomers containing a crosslinkable side group, relative to the total amount of polymer having side groups —CO — NR ¹ R ² .

15. A biosensor according to one of embodiments 1-14, wherein said polymer has a chemical structure represented by formula (III), (IIIa), (IIIb) and / or (IIIc):

where n is from 0.01 to 0.99, am is from 0.99 to 0.01, and, in particular, the sum of m and n is equal to 1; and where R ⁵ is another polymer or diffusion membrane molecule.

16. A biosensor according to one of embodiments 1-15, also comprising a diffusion membrane, wherein the biocompatible layer is the outermost layer.

17. A biosensor according to one of embodiments 1-16, also comprising a diffusion membrane, said diffusion membrane comprising a hydrophilic polyurethane polymer.

18. Biosensor according to one of the options 1-17, which is implantable, in particular implantable subcutaneously.

19. A biosensor according to one of embodiments 1-18, wherein said analyte is an analyte having a molecular weight of less than 1000 Da.

20. Biosensor according to one of options 1-19, in which said analyte is selected from the list consisting of glucose, malate, ethanol, ascorbic acid, cholesterol, glycerin, urea, 3-hydroxybutyrate, lactate, pyruvate, ketones and creatinine, in particular the analyte is glucose.

21. A biosensor according to one of embodiments 1-20, wherein said analyte determination is a living body (in vivo) determination of said analyte.

22. A biosensor according to one of the options 1-21, in which the specified determination of the analyte is continuous monitoring of glycemia.

23. A method of manufacturing an implantable biosensor for analyte determination, comprising at least partial coating of said biosensor with a biocompatible layer, wherein the biocompatible layer comprises a polymer having side groups —CO — NR ¹ R ² , where R ¹ and R ^{2 are} independently selected from —H and C ₁ -C ₆ -alkyl, in particular where at most one of R ¹ and R ² is —H.

24. The method of embodiment 23 wherein R ¹ and R ^{2 are} independently selected from —H and C ₁ -C ₃ alkyl.

25. The method of embodiment 23 or 24, wherein R ¹ and R ^{2 are} independently selected from —H, ethyl and methyl.

26. The method of one of embodiments 23-25, wherein at least one of R ¹ and R ² is methyl.

27. A method according to one of embodiments 23-26, wherein R ¹ and R ² are methyl.

28. A process according to one of embodiments 23-27, wherein said polymer having pendant groups —CO — NR ¹ R ² is obtained or obtained from a composition containing monomers having general structure (I):

where R ³ is a polymerizable group, in particular selected from vinyl, propenyl and isopropenyl, in particular representing vinyl.

29. The method according to one of embodiments 23-28, wherein said polymer having pendant groups —CO — NR ¹ R ² is obtained or obtained from a composition containing mono-N- and / or di-N-substituted acrylamide monomers, in particular containing N, N-dimethylacrylamide monomers.

30. A method according to one of embodiments 23-29, wherein said polymer having pendant —CO — NR ¹ R ² groups is a copolymer, in particular a random copolymer.

31. A method according to one of embodiments 23-30, wherein said polymer having side groups —CO — NR ¹ R ² is a copolymer of mono-N- or di-N-substituted acrylamide monomers and / or O-substituted methacrylate monomers ...

32. The method of embodiment 31, wherein said O-substituted methacrylate monomers are 4-benzoylphenylmethacrylate monomers.

33. A method according to one of embodiments 23-32, wherein said polymer has a chemical structure represented by formula (II) or (IIa):

where R ⁴ is a cross-linkable side group, n is from 0.01 to 0.99, am is from 0.99 to 0.01, in particular the sum of m and n is 1.

34. A method according to one of embodiments 23-33, wherein n is from 0.5 to 0.99, m is from 0.5 to 0.01, and the sum of m and n is 1.

35. The method according to one of embodiments 23-34, wherein said polymer having pendant groups —CO — NR ¹ R ² is a copolymer obtained or obtained from a composition comprising the monomers described in embodiment 28 or 29 and monomers, containing a cross-linkable side group, in particular a benzophenone side group.

36. The method according to one of options 23-35, in which the specified polymer having side groups -CO-NR ¹ R ² contains from 1 to 50 mol. %, in particular from 2 to 25 mol. %, in particular from 3 to 10 mol. %, of units derived from monomers containing a crosslinkable side group, relative to the total amount of polymer having side groups —CO — NR ¹ R ² .

37. A method according to one of embodiments 23-36, wherein said polymer has a chemical structure represented by formula (III), (IIIa), (IIIb) and / or (IIIc):

where n is from 0.01 to 0.99, and m is from 0.99 to 0.01, in particular, the sum of m and n is equal to 1.

38. A method according to one of embodiments 1-37, further comprising at least partially covering said biosensor with a diffusion membrane, wherein the biocompatible layer is the outermost layer.

39. The method of embodiment 38, wherein said diffusion membrane comprises a hydrophilic polyurethane polymer.

40. Use of a biocompatible layer for the manufacture of an implantable biosensor, wherein the biocompatibility layer comprises a polymer having side groups —CO — NR ¹ R ² , where R ¹ and R ^{2 are} independently selected from —H and C ₁ -C ₆ -alkyl, in in particular, where at most one of R ¹ and R ² is —H.

41. The use of embodiment 40 wherein R ¹ and R ^{2 are} independently selected from —H and C ₁ -C ₃ alkyl.

42. The use of embodiment 40 or 41 wherein R ¹ and R ^{2 are} independently selected from —H, ethyl and methyl.

43. Use according to one of embodiments 40-42, wherein at least one of R ¹ and R ² is methyl.

44. Use according to one of Embodiments 40-43, wherein R ¹ and R ² are methyl.

45. A use according to one of embodiments 40-44, wherein said polymer having pendant groups —CO — NR ¹ R ² is obtained or obtained from a composition containing monomers having general structure (I):

where R ³ is a polymerizable group, in particular selected from vinyl, propenyl and isopropenyl, in particular representing vinyl.

46. The use according to one of embodiments 40-45, wherein said polymer having pendant groups —CO — NR ¹ R ² is obtained or obtained from a composition containing mono-N- and / or di-N-substituted acrylamide monomers, in particular containing N, N-dimethylacrylamide monomers.

47. Use according to one of embodiments 40-46, wherein said polymer having pendant groups —CO — NR ¹ R ² is a copolymer, in particular a random copolymer.

48. Use according to one of embodiments 40-47, wherein said polymer is a linear polymer.

49. Use according to one of embodiments 40-48, wherein said polymer having pendant groups —CO — NR ¹ R ² is a copolymer of mono-N- or di-N-substituted acrylamide monomers and / or O-substituted methacrylate monomers ...

50. Use according to embodiment 49, wherein said O-substituted methacrylate monomers are 4-benzoylphenylmethacrylate monomers.

51. Use according to one of embodiments 40-50, wherein said polymer has a chemical structure represented by formula (II) or (IIa):

where R ⁴ is a cross-linkable side group, n is from 0.01 to 0.99, am is from 0.99 to 0.01, in particular the sum of m and n is 1.

52. Use according to one of embodiments 40-51, wherein n is from 0.5 to 0.99, m is from 0.5 to 0.01, and the sum of m and n is 1.

53. Use according to one of embodiments 40-52, wherein said polymer having pendant groups —CO — NR ¹ R ² is a copolymer obtained or obtained from a composition comprising the monomers described in embodiment 45 or 46 and monomers, containing a cross-linkable side group, in particular a benzophenone side group.

54. The use according to one of options 40-53, in which the polymer having pendant groups -CO-NR ¹ R ² contains from 1 to 50 mol. %, in particular from 2 to 25 mol. %, in particular from 3 to 10 mol. %, of units derived from monomers containing a crosslinkable side group, relative to the total amount of polymer having side groups —CO — NR ¹ R ² .

55. Use according to one of embodiments 40-54, wherein said polymer has a chemical structure represented by formula (III), (IIIa), (IIIb) and / or (IIIc):

where n is from 0.01 to 0.99, and m is from 0.99 to 0.01, in particular, the sum of m and n is equal to 1.

56. The use of a biosensor according to one of options 1-55 for continuous determination of an analyte under physiological conditions (in situ).

57. A method for continuous determination of an analyte in a subject's body, characterized in that the determination of said analyte is carried out by means of a biosensor according to one of options 1-22.

58. A method for continuous determination of an analyte in a subject's body, comprising:

(a) implanting a biosensor according to one of options 1-22 into at least one tissue of a subject; and

(b) determination of the specified analyte by means of the specified sensor.

59. A method according to embodiment 57 or 58, characterized in that said continuous determination is carried out by means of the same biosensor for at least one week, in particular at least two weeks, in particular at least three weeks, in particular for at least four weeks.

All sources of information cited in this description are included in it by reference with respect to their entire content and specific information specified in this description.

Description of drawings

FIG. 1a: Results of a study of protein adsorption in experiments with piezoquartz microweighing (PCMW). The graphs show the change in frequency (Δf, Hz) over time (min); The times of protein addition and the times of the buffer wash (s) are indicated by arrows. The lower curves at 20 minutes are curves representing experiments using p (BUMA / HEMA / HPMA) - a copolymer of butyl methacrylate, hydroxyethyl methacrylate and hydroxypropyl methacrylate, and the upper curves at 20 minutes are curves representing experiments using p (DMAA) - copolymer of dimethylacrylamide and benzophenone methacrylate. The test proteins were albumin (in graph A) and fibrinogen (in graph B).

FIG. 16: Results of a study of the adsorption of proteins on polystyrene (PS) in experiments with PCMV. The test proteins were albumin (in graph A) and fibrinogen (in graph B).

FIG. 2: Results of cell adhesion tests carried out on a defunctionalized glass surface (-), on a glass substrate functionalized with polymer p (BUMA / HEMA / HPMA), on a glass substrate functionalized with cross-linked (crosslinked) polymer p (DMAA), and on polystyrene (PS) after a 20-day incubation period. For shooting, a 2.5x (2.5x) or 20x (20x) magnification was used. The cells, visible at 20x magnification in the case of polymer p (DMAA), are cell clumps not adhered to the substrate.

FIG. 3: Percentage cell growth relative to the control (untreated medium) determined by the 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium test (MTT test) or the 2,3-bis test - (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carboxanilide (XTT test), after 24-hour incubation with extracts from samples coated with polymer p (DMAA), samples coated with polymer p (BUMA / HEMA / HPMA), or latex pieces. An untreated culture (nutrient) medium was used as a 100% control sample. Latex pieces were used as a positive control and uncoated polyethylene terephthalate (PET) substrates were used as a negative control.

FIG. 4: Relative cytokine production by THP-1 cells in contact with fibrinogen, activated polyethylene terephthalate (PET) coated with polymer p (BUMA / HEMA / HPMA), or polyethylene terephthalate (PET) coated with polymer p (DMAA). Uncoated PET was used as a negative control, and fibrinogen applied directly to PET as a control (100%). PET substrates directly coated with fibrinogen were used as a control (100%).

Examples are given below, which only illustrate the invention. They should not be construed in any way that limits the scope of protection of the invention.

Reagents used in the Examples

p (BUMA / HEMA / HPMA): copolymer of hydroxyethyl methacrylate, hydroxypropyl methacrylate and butyl methacrylate, taken in a molar ratio of 90: 5: 5; PS: polystyrene; p (DMAA): copolymer of 97% dimethylacrylamide and 3% benzophenone methacrylate; latex: ACCUTech LOT 1307530704; sodium phosphate buffer (PBS) - Dulbecco's buffer solution without calcium or magnesium, manufactured by Lonza (BE17-512F), composition: 0.2 g / l KCl, 0.2 g / l KH ₂ PO ₄ , 8.0 g / l NaCl, 2.16 g / l Na ₂ HPO ₄ ⋅ 7 H ₂ O; sterile physiological saline solution (SPSS).

Protein solutions: albumin solution: 10 mg / ml in PBS buffer; fibrinogen solution: 0.5 mg / ml in a mixture of sodium phosphate buffer (PBS) and sterile physiological saline (SPSS) at a ratio of PBS: SPSS = 1: 1.

Cells and culture medium for their growth: L-929 fibroblast cells (ATCC® CCL-1 ™) were maintained in RPMI 1640 GlutaMAX medium (Gibco 72400-021) supplemented with 10% fetal calf serum ETS (Sigma F7524), 1% sodium pyruvate (Gibco 11360-070) and 1% penicillin-streptomycin (Gibco 15140-122).

XTT test: The cell density was adjusted to approximately 10 ⁵ / ml; 100 μl of this suspension was used for each well of a 96-well plate.

Example 1: Adsorption of Proteins

Protein adsorption to polymer layers was studied using piezoquartz microweighing (PQMW) experiments using a Q-Sense QSX301 device. In these experiments, PCMW substrates (manufactured by Q-Sense) were first coated with a polymer or cross-linked polymer. The coated samples were placed in a measuring cell and vibrated. The resonant vibration frequency was recorded. Then the surface of the samples was covered with a buffer PBS solution, and the polymer coating began to swell until it reached an equilibrium state, as a result of which the resonant vibration frequency ceased to change. The protein adsorption experiment was then started by recording the vibration frequency again and recording the final resonant vibration frequency in the buffer solution. After about 15 minutes, the surface of the sample was again coated with the buffer solution, the protein solution was added to the measuring cell and left covering the surface of the sample for about 15 minutes, after which this surface was again washed with the buffer solution to remove unattached proteins. Proteins adsorbed on the sample surface led to a decrease in frequency, and this decrease correlates with the amount of adsorbed proteins. For our experiments, polymer p (BUMA / HEMA / HPMA) was applied directly to the surface of a PCMV substrate. To functionalize the substrate with a network polymer p (DMAA), a layer of polystyrene was first applied to the substrate, onto which polymer p (DMAA) was centrifuged, which was then fixed in the form of a network structure by UV irradiation.

The experimental results for protein adsorption for polymer p (BUMA / HEMA / HPMA) and cross-linked polymer p (DMAA) are shown in FIG. 1. The addition of albumin solution to the surface resulted in a significant decrease in frequency for the surface coated with polymer p (BUMA / HEMA / HPMA). Most of this decrease could be reversed by rinsing the surface with buffer, but the frequency did not return to the initial level, which indicates that a small amount of protein was irreversibly adsorbed on the surface covered with polymer p (BUMA / HEMA / HPMA). A much smaller decrease in frequency was observed after the addition of albumin solution to the surface of polymer p (DMAA), and this decrease could be completely reversed by washing with buffer. For fibrinogen adsorption, the frequency decrease caused by the protein solution on the surface coated with polymer p (BUMA / HEMA / HPMA) could not be recovered by washing with buffer, and the adsorbed proteins caused a 2 Hz frequency decrease. On the other hand, similar to what was observed with albumin, the vibration frequency of the sample coated with polymer p (DMAA) decreased only slightly after exposure to the surface of the protein solution. It was possible to return this frequency back to the initial level by removing proteins with a buffer. Thus, reticulated polymer p (DMAA) showed improved resistance to adsorption of albumin as well as fibrinogen.

Example 2: Cell adhesion

Cell adhesion was examined on glass cover plates used as substrates. The cover plates consisted of quartz glass and were circular in shape with a diameter of 1.5 cm (VWR, ECN631-1579). Between the respective steps of the experiment, cover plates were kept one at a time in 24-well plates with the cover facing up and in a low dust and low microbial environment. Cover plates were operated with vacuum forceps. The surface of the coverslips was coated with polymer p (BUMA / HEMA / HPMA) by centrifugation, applying the polymer directly to the surface; for polymer p (DMAA) coating, the surface of the cover plates was first spun coated with polystyrene (PS), then spun coated with polymer p (DMAA), again followed by UV irradiation (365 nm, specific power 0, 35 W / m ^2, for 2 min). Uncoated and only polystyrene coated coverslips were used as controls.

To avoid microbial contamination of cells, coated coverslips were sterilized by immersion for 15 seconds in 70% ethanol in water or Perform solution (25% ethanol, 35% propan-1-ol, and 40% water) and the fact that the cover plates were operated in a sterile casing. After drying the sterilization solution, cover plates were placed in the wells of a 24-well plate containing 500 μl of culture medium. Air bubbles were removed from under the coverslips by pressing the coverslips to the bottom of the wells with tweezers. The coated cover plates were allowed to swell in the culture medium for approximately one hour prior to use.

L929 cells were grown in a nutrient medium in a flask or a T75 flask to a population of 90%. The cells were separated from the culture flask by washing with Dulbecco's sodium phosphate buffer solution followed by the addition of 1 ml of trypsin / EDTA mixture. The cells were trypsinized for approximately five minutes at 37 ° C, after which 9 ml of medium was added. The cells were carefully resuspended and centrifuged for two minutes with an overload of 500 g. After centrifugation, the cell clot was resuspended in 5 ml of fresh medium (first with a 10 ml serological pipette, followed by resuspension with an Eppendorf pipette 1 ml). The cell density was adjusted by diluting 2 ml of resuspended cells with 73 ml of culture medium; from the suspension thus adjusted, 1 ml portions were placed on the cover plates, so that approximately 30,000 cells were inoculated into each well. After seeding, the cells were grown in a standard carbon dioxide (CO ₂ ) incubator.

The results of this experiment after a 20 day incubation period are shown in FIG. 2. As can be seen in the above images, the defunctionalized surface of the glass substrate was completely, for example, in a continuous manner, covered with a layer of cells, and this indicates that this surface does not have resistance to cell adhesion. Cells failed to adhere to the surface coated with polymer p (BUMA / HEMA / HPMA) after the first 3 days. After a longer incubation period, some cells were able to successfully and irreversibly attach to the surface and began to grow and multiply. After 20 days, most of the surface coated with polymer p (BUMA / HEMA / HPMA) was coated with cells (FIG. 2). On the surface coated with reticulated polymer p (DMAA), no significant cell adhesion was observed. After 20 days, the cells were able to attach only to each other with the formation of cell clumps. By carefully moving the samples, it was observed that the cell clumps floated freely and could not attach to the surface covered with reticulated polymer p (DMAA). The results of this experiment demonstrated that the surface coated with reticulated polymer p (DMAA) has a strong resistance to cell adhesion.

Example 3: Toxicity Test

For the toxicity test, both sides of a PET backing (2x2 cm) were coated with polymer p (DMAA) reticulated polymer p (DMAA) by centrifugation followed by UV exposure. Before use, the samples were sterilized by the electron beam method (absorbed dose 25 kGy). Three test samples were immersed in 4 ml of culture medium for 24 hours at 37 ° C. 1 ml of the incubated culture medium (eluate) was transferred into a well of a fresh 96-well plate, fibroblast cells of the L929 line were added and incubated for another 24 hours, after which the percentage of surviving cells was measured using the XTT test. Results are presented as percentage of cell growth, with untreated culture medium being used as a control (eg 100%). Thus, the lower the percentage growth value, the stronger the toxic effect of the sample in the cells. Pieces cut from latex gloves and eluates prepared as above from PET substrates coated with copolymer p (BUMA / HEMA / HPMA) were used as controls. For example, pieces cut from latex gloves were used as a positive control, and uncoated PET substrates were used as a negative control. As shown in FIG. 3, p (DMAA) coating showed no detectable toxicity on L929 cells.

Example 4: Activation of Cells with Fibrinogen Coated Polymers

The human cell line THP-1 was used to analyze the proinflammatory potential of the polymers. THP-1 cells are monocytic cells that can be activated by exogenous influencing factors that cause cells to increase the secretion of cytokines into the culture medium. Such activation can be induced by direct surface contact with an inducing material or soluble components released from such material. Another possibility is the activation of cells by fibrinogen, activated by contact with the polymer surface. This activation of fibrinogen results in a change in the conformation-exhibiting binding sites that bind to immune receptors on THP-1 cells, thereby causing cell activation. Another possibility is the activation of cells on the surface of the coating due to the adsorption of fibrinogen. This attachment to the surface causes a conformational change in fibrinogen, whereby the binding sites are exposed and able to bind to immune receptors on THP-1 cells, thereby causing cell activation.

In this experiment, small sheet-like polyethylene terephthalate (PET) substrates were coated on one side with a polymer (p (BUMA / HEMA / HPMA) or p (DMAA)). The coating of the substrates with fibrinogen was performed by adding a fibrinogen solution at a concentration of 2 mg / ml to the substrates and incubating at 37 ° C for 16 hours. Excess fibrinogen solution was removed and THP-1 cells were added. After 48-hour incubation at 37 ° C in an incubator, the supernatant was separated from the cells and cytokines released into the supernatant were analyzed. Cytokine analysis was performed using Cytometric Bead Array (CBA) assays from BD Biosciences (Human Chemokine Kit and Human Inflammatory Cytokine Kit, both according to the manufacturer's instructions). Fibrinogen-only coated substrates were used as controls.

The cytokine levels observed on the surfaces of the polymers p (BUMA / HEMA / HPMA) and p (DMAA) were normalized to the cytokine levels on the surface of the control and are shown in FIG. 4. Samples coated with polymers p (BUMA / HEMA / HPMA) and p (DMAA) showed comparable results in the activation of human interleukin-1β (IL-1β), human tumor necrosis factor (TNF) and human interleukin-8 (IL- eight). In addition, the cytokine production of monocyte chemoattract protein-1 (MCP-1), human vascular endothelial growth factor (VEGF) was slightly lower on the surface of polymer p (DMAA) than on the surface of polymer p (BUMA / HEMA / HPMA). Interestingly, a significantly lower level of human interferon-γ inducible protein (IP-10) was shown on the surface of polymer p (DMAA) than on the surface of polymer p (BUMA / HEMA / HPMA). The results of this experiment show that, compared to polymer p (BUMA / HEMA / HPMA), the surface coated with reticulated polymer p (DMAA) exhibits increased biocompatibility.

List of cited literature

US 2006/0198864 A1

EP 0821234

EP 0974303

US 2005/0023152

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WO 2007/012494

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WO 2011/012269

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WO 2015/005953 A1

J. Hones et al. The Technology Behind Glucose Meters: Test Strips, Diabetes Technology & Therapeutics, Volume 10, Supplement 1, 2008, pp. 10-26.

Claims (21)

1. A biosensor for determining an analyte comprising a sensor module at least partially covered with a biocompatible layer, the biocompatible layer comprising a polymer having side groups —CO — NR ¹ R ² , where R ¹ and R ^{2 are} independently selected from —H and C ₁ -C ₆ -alkyl, and having a chemical structure represented by formula (II) or (IIa):

where R ⁴ is a cross-linkable or cross-linked side group, n is from 0.01 to 0.99, am is from 0.99 to 0.01, in particular, the sum of m and n is 1. 2. Biosensor according to claim 1, in which R ¹ and R ^{2 are} independently selected from —H and C ₁ -C ₃ -alkyl, in particular R ¹ and R ^{2 are} independently selected from methyl, —H and ethyl, in particular R ¹ and R ² are methyl. 3. A biosensor according to claim 1 or 2, wherein said polymer having pendant groups —CO — NR ¹ R ² is derived or derived from a composition comprising monomers having the general structure (I):

where R ³ is a polymerizable group, in particular selected from vinyl, propenyl and isopropenyl, in particular representing vinyl. 4. Biosensor according to one of paragraphs. 1-3, wherein said polymer having pendant —CO — NR ¹ R ² groups is a random copolymer. 5. Biosensor according to one of paragraphs. 1-4, wherein said polymer having pendant groups —CO — NR ¹ R ² is a copolymer of mono-N- or di-N-substituted acrylamide monomers and / or O-substituted methacrylate monomers, in particular, said O-substituted methacrylate monomers are 4-benzoylphenylmethacrylate monomers. 6. Biosensor according to one of paragraphs. 1-5, in which n is from 0.5 to 0.99, m is from 0.01 to 0.5, and the sum of m and n is 1. 7. Biosensor according to one of paragraphs. 1-6, in which said polymer has a chemical structure represented by formula (III), (IIIa), (IIIb) and / or (IIIc):

where n is from 0.01 to 0.99, am is from 0.99 to 0.01, and, in particular, the sum of m and n is equal to 1; and where R ⁵ is another polymer or diffusion membrane molecule. 8. Biosensor according to one of paragraphs. 1-7, also comprising a diffusion membrane, said diffusion membrane comprising a hydrophilic polyurethane polymer. 9. Biosensor according to one of paragraphs. 1-8, which is implantable, in particular implantable subcutaneously. 10. Biosensor according to one of paragraphs. 1-9, in which the specified analyte is selected from the list consisting of glucose, malate, ethanol, ascorbic acid, cholesterol, glycerol, urea, 3-hydroxybutyrate, lactate, pyruvate, ketones and creatinine, in particular the analyte is glucose, and the specified analyte determination is, in particular, continuous monitoring of glycemia. 11. A method of manufacturing an implantable biosensor for determining an analyte, comprising at least partial coating of said biosensor with a biocompatible layer as described in one of claims. 1-7. 12. The use of providing biocompatibility layer, characterized in one of the paragraphs. 1-7, for the manufacture of an implantable biosensor. 13. A method for continuous determination of an analyte in a subject's body, characterized in that the determination of said analyte is carried out by means of a biosensor according to one of claims. 1-10. 14. The method according to claim 13, characterized in that said continuous determination is carried out by means of the same biosensor for at least one week, in particular at least two weeks, in particular at least three weeks, in particular at least at least four weeks.

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